Local anesthetic lidocaine-inducible gene, growth differentiation factor-15 suppresses the growth of cancer cell lines

Administration of local anesthetics, such as lidocaine, in the perioperative period improves outcomes of cancer patients. However, its precise mechanism is still unresolved. The growth of human cancer cell lines, including HeLa cells, are suppressed by lidocaine treatment. We identified that growth differentiation factor-15 (GDF-15) was commonly upregulated in lidocaine-treated cancer cell lines. GDF-15 is a divergent member of the transforming growth factor-β (TGF-β) superfamily and it is produced as an unprocessed pro-protein form and then cleaved to generate a mature form. In lidocaine-treated HeLa cells, increased production of GDF-15 in the endoplasmic reticulum (ER) was observed and unprocessed pro-protein form of GDF-15 was secreted extracellularly. Further, lidocaine induced apoptosis and apoptosis-inducible Tribbles homologue 3 (TRIB3) was also commonly upregulated in lidocaine-treated cancer cell lines. In addition, transcription factor C/EBP homologous protein (CHOP), which is a positive regulator of not only GDF-15 but TRIB3 was also induced by lidocaine. Lidocaine-induced growth suppression and apoptosis was suppressed by knockdown of GDF-15 or TRIB3 expression by small interference RNA (siRNA). These observations suggest that lidocaine suppresses the growth of cancer cells through increasing GDF-15 and TRIB3 expression, suggesting its potential application as cancer therapy.


Growth of cancer cell lines was suppressed by lidocaine treatment.
To examine the effect of the amide-linked local anesthetic, lidocaine, on the growth of human cancer cell lines, colony formation assay was performed using cervical cancer HeLa, osteosarcoma U2OS, hepatocellular carcinoma HepG2 and colon cancer SW480 cells. Ropivacaine, which is also an amide-linked local anesthetic, was previously shown to inhibit the proliferation of HepG2 cells and doxorubicin-resistant U2OS cells 33,34 . It also suppressed metastasis of colon cancer SW620 cells 12 . Furthermore, combination therapy with ropivacaine and nutrient restriction suppressed the growth of HeLa cells 35 . Thus, we examined the effects of lidocaine on these and similar cell lines. The growth of HeLa cells was significantly suppressed by 1 mM lidocaine treatment. The growth of U2OS and HepG2 cells was suppressed by lower lidocaine concentrations of 0.1 mM and 0.3 mM, respectively, compared with that of HeLa cells. Conversely, the growth of SW480 cells was effectively suppressed by a higher lidocaine concentration of 3 mM. These results indicate that the growth of these cell lines was inhibited by lidocaine in a concentrationdependent manner, with the cell lines showing differential sensitivities to lidocaine-induced growth suppression ( Fig. 1a-d).
Comprehensive transcriptome sequence analysis of lidocaine-treated cancer cell lines. To elucidate the mechanism of lidocaine-induced growth suppression of cancer cell lines, we examined the gene expression profile of the four cell lines with and without lidocaine treatment. HeLa, U2OS, SW480 and HepG2 cells were treated with the different concentrations of lidocaine, found to result in induction of significant growth inhibition (1 and 3 mM for HeLa and U2OS cells, 1 and 5 mM for SW480 cells and 0.5 and 1 mM for HepG2 cells). After treatment for 24 h, total RNAs were purified from the cells and high-quality RNA samples were used for RNA-seq using NextSeq500 (Illumina Inc.) as described in the Methods. Differentially expressed genes (DEGs) between two conditions were detected using TCC-iDEGES-DESeq pipeline on R (https:// www.R-proje ct. org/, version 4.0.3) package TCC (version 1.26.0). Genes with a false discovery rate (FDR, adjusted p value) of < 0.05 were defined as being significant DEGs. Two hundred eighty-six genes were extracted as DEGs on at least one analysis (Fig. 2a). In addition, we identified the commonly modified genes in the examined cancer cell lines, and found that the expression levels of nine genes were significantly changed in response to lidocaine ( Fig. 2b and Table 1). Among these genes, GDF-15 was upregulated in a lidocaine dose-dependent manner, suggesting that GDF-15 plays a significant role in lidocaine-induced growth suppression of cancer cell lines.

Lidocaine increased expression and secretion of pro-protein GDF-15.
To confirm the increased expression of GDF-15, qPCR was performed using lidocaine-treated HeLa cells. Lidocaine induced GDF-15 expression in a concentration-dependent manner and the GDF-15 level significantly increased by about 45-fold with 3 mM lidocaine treatment compared with that in untreated HeLa cells (Fig. 3a). We next examined the levels of secreted GDF-15 by enzyme-linked immunosorbent assay (ELISA) using the culture medium of lidocainetreated HeLa cells. After normalization of GDF-15 levels by cell number, it was revealed that lidocaine increased the extracellular secretion of GDF-15. Treatment with 5 mM lidocaine almost completely suppressed the growth of HeLa cells, although the secretion of GDF-15 increased about eightfold, suggesting that lidocaine-induced growth inhibition correlates with increased secretion of GDF-15 (Fig. 3b). We further performed Western blotting using GDF-15 polyclonal antibodies purified from rabbits immunized with GDF-15 peptide (30-308 aa). The use of cell lysates and culture medium treated with lidocaine resulted in an increase of pro-protein GDF-15 (34 kDa) both intracellularly and in the culture medium (Fig. 3c). These results indicated that lidocaine-induced www.nature.com/scientificreports/ GDF-15 was at least pro-protein form and was secreted extracellularly in a concentration-dependent manner. In addition, we investigated the cellular distribution of lidocaine-induced GDF-15. Immunofluorescence analysis showed that GDF-15 was localized both in the nucleus and cytoplasm in lidocaine-untreated HeLa cells. After treatment with 3 mM lidocaine, increased GDF-15 was observed around the perinuclear region. Co-staining of  www.nature.com/scientificreports/ the ER chaperon glucose-regulated protein 78 (GRP78) revealed that increased GDF-15 was localized in the ER. In addition, the expression level of GRP78 was increased in lidocaine-treated HeLa cells in immunofluorescence assay although its level of mRNA was not increased in RNA-seq analysis, indicating that GRP78 was upregulated at translational level after lidocaine treatment (Fig. 3d, Supplementary Table 1). Furthermore, increased GDF-15 accumulated in the pericellular region in lidocaine-treated HeLa cells (Fig. 3d). These results demonstrate that lidocaine induced the expression and secretion of pro-protein form of GDF-15.

Lidocaine-induced growth inhibition is suppressed by knockdown of pro-protein GDF-15.
We Lidocaine induces apoptosis and TRIB3 expression. Since lidocaine caused growth suppression in cancer cell lines, we examined whether apoptosis was induced in lidocaine-treated HeLa cells. We observed that apoptotic cells with a condensed chromosome were hardly observed in lidocaine-untreated HeLa cells, while such cells increased in lidocaine-treated cells in a dose-dependent manner (Fig. 5a). RNA-seq revealed that apoptosis-inducible TRIB3 was also significantly upregulated as a commonly modified gene in the examined cancer cell lines with lidocaine treatment (Fig. 2b, Table 1). qPCR analysis showed that TRIB3 expression in HeLa cells treated with 3 mM lidocaine significantly increased by approximately 35-fold compared with that in untreated HeLa cells (Fig. 5b). In addition, transcription factor CHOP transactivates not only GDF-15 but also TRIB3 expression and lidocaine-treated HeLa and U2OS cells exhibited the tendency that CHOP expression was increased in a lidocaine-concentration dependent manner (Supplementary Table 1) 27,32 . qPCR showed that CHOP expression was increased in a lidocaine-concentration dependent manner and its expression in HeLa cells treated with 3 mM lidocaine was upregulated by approximately sevenfold compared with that in untreated HeLa cells, indicating that CHOP is involved in lidocaine-induced gene expression in HeLa cells (Fig. 5c).

Lidocaine-induced apoptosis is suppressed by TRIB3 knockdown. Although lidocaine-induced
growth suppression was effectively inhibited by GDF-15 knockdown, growth suppression was not completely inhibited. Thus, we examined the expression of TRIB3 in GDF-15 knockdown HeLa cells. TRIB3 expression increased with lidocaine treatment in both control and GDF-15 siRNA-transfected HeLa cells (Fig. 6a). To examine whether lidocaine-induced TRIB3 plays a role in the induction of apoptosis, we performed TRIB3 knockdown experiment. Western blotting using HeLa cells transfected with control siRNA showed an increase in TRIB3 expression after lidocaine treatment. On the other hand, TRIB3 expression was suppressed in HeLa cells transfected with TRIB3 siRNA with and without lidocaine treatment (Fig. 6b). We next examined TRIB3 expression by immunofluorescence assay. TRIB3 was expressed in the nucleus and its expression increased after lidocaine treatment in HeLa cells transfected with control siRNA, while its expression in the nucleus was suppressed in lidocaine-treated HeLa cells transfected with TRIB3 siRNA (Fig. 6c). Apoptotic cells were also increased in control siRNA transfected HeLa cells after lidocaine treatment. However, the number of lidocaineinduced apoptotic cells decreased in HeLa cells in which TRIB3 expression was suppressed by siRNA compared with HeLa cells transfected with control siRNA, indicating that lidocaine-induced TRIB3 plays a role in the www.nature.com/scientificreports/ www.nature.com/scientificreports/ induction of apoptosis (Fig. 6d). Taken together, lidocaine-induced pro-protein form of GDF-15 plays a role in growth suppression in HeLa cells, which accompanied with expression of apoptosis-inducible TRIB3 (Fig. 7).

Discussion
In this study, we demonstrated that lidocaine induces growth suppression of cancer cell lines through increasing of GDF-15 expression. Additionally, lidocaine-induced GDF-15 was revealed to be pro-protein form of GDF-15 (pro-GDF-15). This form is known to be more rapidly secreted than the mature form 36 . Normal thyroid cells express pro-GDF-15, whereas papillary thyroid tumors express mature GDF-15 in large amounts 37 . Furthermore, decrease of pro-GDF-15 stored in the stroma of prostate cancer cells leads to poor outcomes after prostatectomy 15 .
In addition, mutation of the pro-peptide region of GDF-15 (N-terminal region of pro-GDF-15) is involved in the poor prognosis of oral squamous cell carcinoma 38 . In our study, the GDF-15 knockdown experiment using siRNAs revealed that decreased pro-GDF-15 suppressed lidocaine-induced growth inhibition of HeLa cells, indicating that lidocaine-induced pro-GDF-15 might play a significant role in tumor suppression. Although GDF-15 has tumor suppressive effects, increased serum GDF-15 in cancer patients indicates advanced disease 39 . A study of the effect of GDF-15 through glial cell-derived neurotrophic factor (GDNF) family receptor α-like protein (GFRAL) demonstrated that increased GDF-15 is related to the anorexia/cachexia observed in advanced cancer patients 40 . On the other hand, the GDF-15 receptor related to tumor suppression is still unidentified. In cervical cancer cell lines, including HeLa cells, GDF-15 promotes proliferation through phosphorylation of ErbB2, a member of the epithelial growth factor (EGF) receptor, and activation of PI3K/ Akt and ERK signaling pathways 41 . Although this appears to be inconsistent with the results of our study, we speculate that the receptor for lidocaine-induced pro-GDF-15 might be different from ErbB2. A study using purified membrane fractions reported that a small fraction of GRP78 exists on the cell surface (CS-GRP78) transmitting Akt-mediated survival signaling and another study reported that monoclonal antibodies against for CS-GRP78 inhibit this survival signaling 42,43 . Lidocaine-induced pro-GDF-15 might have the potential to modify CS-GRP78-Akt-mediated survive signal. In addition, increased pro-GDF-15 in the ER might prevent GRP78 translocation from the ER to the cell surface.
In cancer cells, unregulated protein production disrupts fundamental ER functions such as protein folding, resulting in a state known as ER stress. Previous study reports that lidocaine causes ER stress-related apoptosis in adrenal pheochromocytoma PC12 cells 44 . Other amide-linked local anesthetics, such as bupivacaine and levobupivacaine inhibit the growth of colon cancer Caco-2 cells with activation of the ER stress response 45 . In addition, lidocaine upregulated CHOP, which is ER stress-related transcription factor induced by activating transcription factor 4 (ATF4) following ER stress sensor PKR-like ER kinase (PERK) activation 31 . Thereby, we examined whether lidocaine induces ER stress in HeLa cells. ER stress sensors such as PERK, inositol-requiring enzyme 1 (IRE1) and activating transcription factor 6 (ATF6) are activated by autophosphorylation or cleavage in ER stress 31 . Although phosphorylation of IRE1 was slightly increased, phosphorylation of PERK was hardly observed in lidocaine-treated HeLa cells ( Supplementary Fig. 4a and b). In addition, ATF6 cleavage was not significantly increased though ATF6 expression was slightly increased in these cells ( Supplementary Fig. 4c). Actually, only a few of ER stress-related molecules was upregulated in RNA-seq using lidocaine-treated cancer cell lines (Supplementary Table 1). Since ER stress is induced with treatment of 10 mM lidocaine in PC12 cells, treatment of such high concentration of lidocaine might be necessary to induce ER stress in cancer cell lines including HeLa cells. On the other hand, Tsai et al. reported that ER stress-inducible isochaihulactone, a natural compound extracted from herb induces apoptosis through induction of DDIT3 (CHOP), which is independent of GRP78 and PERK expression in glioblastoma multiform cells, indicating the existence of CHOP-mediated apoptosis pathway which is independent of the canonical ER stress signaling 46 . Further experiments are necessary to verify lidocaine-inducible ER stress in cancer cells.
Capsaicin-induced TRIB3 expression causes apoptosis through preventing AKT activity by inhibition of its phosphorylation 47 . In addition, capsaicin-induced GDF-15 causes apoptosis in colon cancer cell lines such as HCT116 cells 48 . These reports suggest that not only GDF-15 and but also TRIB3 is important in regulating the proliferation of cancer. Although, we could not elucidate the precise correlation between GDF-15 and TRIB3 in growth inhibition or apoptosis of HeLa cells, further experiments including other commonly modified genes should be performed to explain the roles of lidocaine in cancer pathology (Fig. 2b, Table 1) 49 .
In conclusion, our study indicates the possibility that locally administered lidocaine for pain control in cancer patients might suppress tumor growth by increasing GDF-15 and TRIB3 expression.

Methods
Cell cultures and reagents. Human cervical cancer HeLa (RCB0007) and hepatocellular carcinoma HepG2 (RBC1648) cells were provided by the RIKEN BioResearch Resource Center (BRC) through the National Bio-Resource Project of MEXT, Japan. Human osteosarcoma U2OS and colon cancer SW480 cells were purchased from American Type Culture Collection (VA, USA). HeLa, HepG2 and U2OS cells were cultured in Dulbecco Modified Eagle medium and SW480 cells were cultured in Leibovitz's L-15 medium (Fujifilm-Wako, Osaka, Japan) supplemented with 10% (v/v) fetal bovine serum (Atlas Biologicals, CO, USA) at 37 °C under 5% CO 2 . Lidocaine hydrochloride isotonic solution (2%w/v) (Aspen, Tokyo, Japan) was added to the culture medium to achieve the test concentrations. PBS was used to adjust the total dose of lidocaine in each sample.
Colony formation assay. Cells cultured in a six-well dish at the concentration of 3 × 10 4 cells per well for two days were treated with lidocaine and cultured for a further 7 days. Cells fixed with 4% paraformaldehyde were stained with 0.5% crystal violet. For quantification of the colony number, the stained cultures dishes were treated with 10% acetic acid and absorbance of the solution was measured at 592 nm. ELISA. HeLa cells were cultured in a twelve-well dish at the concentration of 4 × 10 4 per well for two days.
Cells were treated with lidocaine at the indicated concentration for 24 h and the culture medium was applied to the human GDF-15 immunoassay kit (Proteintech, Rosemont, IL, USA) following the manufacturer's instructions. Samples were incubated with anti-GDF-15 antibodies coated on a microplate for 2 h at 37 °C, following which the wells were washed four times. Next, the wells were incubated with GDF-15 antibody conjugated with horseradish peroxidase for 1 h at room temperature. After washing, the wells were incubated with 3,3' ,5,5'-tetramethyl-benzidene (TMB) substrate solution for 30 min at room temperature. After addition of the sulfuric acid stop solution, the intensity of the solution in each well was measured at 450 nm.    Fig. 4a-c). A primary antibody reaction using Can Get Signal Immunoreaction Enhancer Solution (NKB101, Toyobo Biotechnology Co. Ltd., Osaka, Japan) was performed for 1 h at room temperature, and the  www.nature.com/scientificreports/ membranes were washed with TBS-T. An HRP-conjugated secondary antibody reaction was performed at room temperature for 1 h. After washing the membranes, Western Lighting Plus (Perkin Elmer, MA, USA) and Merstham Imager 600 (Cytiva, Tokyo, Japan) were used for protein detection.

Antibodies.
Immunofluorescence assay. HeLa cells were cultured on cover glasses. The cells were fixed with 4% paraformaldehyde for 30 min and permeabilized with 0.2% Triton-X for 5 min at room temperature. 5% bovine serum albumin (BSA) was used for blocking for 60 min at room temperature. The primary antibody was incubated for 60 min at room temperature and washed three times with PBS. Fluorescence conjugated secondary antibody was incubated for 60 min at room temperature and washed three times with PBS. DNA was stained with 300 nM 4' ,6-diamidino-2-phenylindole (DAPI) (Invitrogen, MA, USA) for 15 min at room temperature. The images were observed by immunofluorescence microscopy (Nikon Eclipse Ni-E, Tokyo, JAPAN) equipped with NIS-Elements Ver 5.1 software.   Lidocaine induced production of pro-protein form of GDF-15 (pro-GDF-15) in the ER and pro-GDF-15 that accumulates in the pericellular region is then secreted extracellularly. Secreted pro-GDF-15 might inhibit the survival signals or transmit apoptotic signals. Lidocaine also induces TRIB3 expression, which has a role in apoptosis.